1. Field of the Invention
The present invention relates generally to the use of hybridization chain reaction for in situ imaging.
2. Description of the Related Art
Hybridization Chain Reaction (HCR) is a novel method for the triggered chain of hybridization of nucleic acid molecules starting from stable, monomer hairpins or other more complicated nucleic acid structures. HCR is described in U.S. patent application Ser. No. 11/087,937, filed Mar. 22, 2005, which is incorporated herein by reference. In the simplest version of this process, stable monomer hairpins undergo a chain reaction of hybridization events to form a nicked helix when triggered by a nucleic acid initiator strand. The fundamental principle behind HCR is that short loops are resistant to invasion by complementary single-stranded nucleic acids. This stability allows for the storage of potential energy in the form of loops; potential energy is released when a triggered conformational change allows the single-stranded bases in the loops to hybridize with a complementary strand. HCR is described in co-pending provisional patent application Ser. No. 60/556,147 filed on Mar. 25, 2004, incorporated herein by reference in its entirety, and one embodiment is illustrated in FIG. 1. A more complex embodiment, quadratic HCR, is shown in FIG. 2.
In situ hybridization methods enable the detailed spatial mapping of genes and mRNAs in normal and pathological tissues, allowing the study of gene expression and regulation in a morphological context from the sub-cellular to the organismal levels. Target nucleic acids are identified via the hybridization of nucleic acid probes that facilitate subsequent imaging by one of a number of methods. Radiolabeled probe molecules provide high sensitivity, but poor spatial resolution and the disadvantage of working with biohazardous materials has motivated the development of several nonradioactive alternatives. In situ biological imaging of an analyte (including not only nucleic acids, but proteins and other molecules and compounds) or multiple analytes can be accomplished by a variety of methods; however, such techniques have limitations, particularly as the number of analytes increases.
Fluorescence in situ hybridization (FISH) methods enable the detailed mapping of transcriptionally active genes from sub-cellular to organismal levels. (Lawrence et al. Cell, 57:493-502, 1989; Kislauskis et al. The Journal of Cell Biology, 123(1):165 172, 1993; Wilkie et al. Current Biology, 9: 1263-1266, 1999; Levsky, et al. Science 297:836-840, 2002; D. Kosman, et al. Science, 305:846, 2004.) Fluorescently-labeled nucleic acid probes are amenable to multiplexing (Levsky, 2002) but provide low sensitivity due to the small number of dyes per probe (Kosman, 2004). Immunological methods employ antibodies to bind haptenated nucleic acid probes which are then detected using fluorescently-labeled secondary antibodies (Kosman, 2004; Hughes et al. BioTechniques, 24(4):530-532, 1998). Some amplification can be achieved by introducing additional layers of labeled antibodies, (P. T. Macechko, et al. J Histochem Cytochem, 45(3):359-363, 1997) but the sensitivity is insufficient for imaging low-abundance mRNAs. Spatially localized signal amplification can be achieved using horseradish peroxidase-labeled antibodies (Wilkie, 1999; Kosman, 2004) or probes (M. P. C. van de Corput et al. J Histochem Cytochem, 46(11):1249-1259, 1998) to catalyze the binding of fluorescent tyramides in the vicinity of the probe. This approach significantly enhances sensitivity, but serial multiplexing results in sample degradation. Similar sensitivities have been achieved using in situ PCR, but the method is more cumbersome and the results are less reproducible. All of the above methods suffer from enhanced background signal due to the nonspecific binding of nucleic acid probes prior to fluorescent labeling or amplification.
Another technique for amplifying a signal from a hybridization event is the branched DNA (bDNA) approach, in which a pre-amplifier strand hybridizes to a portion of the probe, which in turn serves as a nucleation site for the hybridization of a fixed number of multiply-labeled amplifier strands. (Schweitzer et al. Curr Opin Biotechnol, 12:21-27, 2001.; Qian et al. Diagnostic Molecular Pathology, 12(1):1-13, 2003; Collinset al.. Nucleic Acids Res, 25(15):2979-2984, 1997.; Bushnell, et al. Bioinformatics, 15(5):348-355, 1999; Player, et al. J Histochem Cytochem, 49(5):603-611, 2001.)